UV-B irradiation-activated E3 ligase GmILPA1 modulates gibberellin catabolism to increase plant height in soybean

Plant height is a key agronomic trait that affects yield and is controlled by both phytohormone gibberellin (GA) and ultraviolet-B (UV-B) irradiation. However, whether and how plant height is modulated by UV-B-mediated changes in GA metabolism are not well understood. It has not been reported that the E3 ubiquitin ligase Anaphase Promoting Complex/Cyclosome (APC/C) is involved in the regulation of plant growth in response to environmental factors. We perform a forward genetic screen in soybean and find that a mutation in Glycine max Increased Leaf Petiole Angle1 (GmILPA1), encoding a subunit of the APC/C, lead to dwarfism under UV-B irradiation. UV-B promotes the accumulation of GmILPA1, which ubiquitinate the GA catabolic enzyme GA2 OXIDASE-like (GmGA2ox-like), resulting in its degradation in a UV-B-dependent manner. Another E3 ligase, GmUBL1, also ubiquitinate GmGA2ox-like and enhance the GmILPA1-mediated degradation of GmGA2ox-like, which suggest that GmILPA1-GmGA2ox-like module counteract the UV-B-mediated reduction of bioactive GAs. We also determine that GmILPA1 is a target of selection during soybean domestication and breeding. The deletion (Indel-665) in the promoter might facilitate the adaptation of soybean to high UV-B irradiation. This study indicates that an evolutionary GmILPA1 variant has the capability to develop ideal plant architecture with soybean cultivars.

3) Clarify that GmUID1 is GmILPA1, and that the Gmuid1-2 has been published before as Gmilpa1 mutant.Line 17: "...H12 named as Gmuid1, the other name Gmilpa1...".Not clear... Shouldn't the name previously given be kept?That would be clearer than having several names for the same gene.Is Gmuid1-2 the previously published Gmilpa1 mutant (Gao et al., Plant Physiol 2017)?Seems like from the description of the mutation (1149-bp deletion incl.4th exon...).Thus why "... we isolated additional mutant allele...".Isolated from where if already published.Please clarify.Lines 181-182: "Sequence comparison and phylogenetic analysis demonstrated that GmUID1 encodes an AP8C-like protein, ...", rather confirmed, if at all, as the gene has already been published before (Gao et al 2017 entitled "GmILPA1, Encoding an APC8-like Protein, Controls Leaf Petiole Angle in Soybean") Line 187: "..., which revealed that GmUID1-GFP localizes to both the nucleus and cytoplasm..."; also confirmed, as already shown by Gao et al., 2017 for GmILPA1, which = GmUID1... 4) Please add western analysis of GmGA2ox-like protein levels in wild-type under UV and no-UV conditions, as well as same for Gmuid1.An anti-GmGA2ox-like antibody is available, but was only used in cell-free degradation assays (Fig. 4i,j).An effect on GmGA2ox-like levels should be tested in western analyses as well.Elevated levels in Gmuid1?Reduced levels in wild type under UV versus no-UV?5) Fig 5c: discuss why +GA only suppresses dwarfism of Gmuid1, but results in strongly changed plant morphology compared to wild type.
-Line 472: Light and growth conditions?How old plants?How long exposed to UV-B? -Fig. 2 b-h.Conditions are not clearly described, but it seems 2b very different to 2d-h.Re-arrange figure that one does not compare plant height in 2b versus others measurements.As they are directly next to each other, it is rather confusing.Mention the light treatments in the legend (duration, fluence rate).
-Fig.4a: why is there such a strong difference in the input between -and + GmGA2ox-like-GFP on total Ub-Flag?-Lines 739 -740: provide fluence rates in umol m-2 sec-1.
Reviewer #3 (Remarks to the Author): Sun et.al. provides a new hypothesis on the post-translational regulation of GA2ox-like in a UVBdependent manner in Soybean.This is an intriguing possibility which was not been explored before.The authors also tried to provide evidence for the E3 ligases that ubiquitinate the GA2ox-like.In addition, the prevalence of different haplotypes in one of the E3s, the GmUID1 has been investigated and proposed that a defect in its cis-element may have contributed to a UVB-dependent plant height phenotype.
Even though the manuscript provides a large amount of experimental data, I have some serious concerns over the ways in which the experiments were conducted and the rationale behind the explanation and interpretation of the results.I have the following major and minor comments that I request the authors to address in detail: Major: 1.The authors provide Gmilpa1 as a mutant allelic to Gmuid1-1.Gmilpa1 has been published previously (ref.53 in the manuscript).However, Gmilpa1 do not show a dwarf phenotype (Figure 1 and 3 of ref. 53).Can the authors please clarify this discrepancy?2.Even though the authors show data for the mutual interaction between GmUID1, GmUBL1 and GmGA2-ox-like, it is not clear how these interactions ultimately cooperate in GmGA2ox-like degradation in presence of UVB.There are several questions remain unanswered: a.What is the role of GmUBL1 in GmUID1-GmGA2ox-like interaction influenced by UVB? b.What are the respective contributions of GmUBL1 and GmUID1 in ubiquitinating GmGA2ox-like?c.What is the relevance of direct interaction between GmUBL1 and GmUID1?
3. The experiments involving different wavelengths of light are not convincing a. UVB is often detrimental to plant growth, so weak narrow-band UVB coupled with white light is preferred, especially at the seedling stage.However, the authors have used intense UVB radiation alone (methods section; Lines 739-741).This could induce potential stress on the plants.Moreover, the exact experimental conditions used are unclear b.For Fig. 2C-2H: The quantity, duration and frequency of light treatment were not mentioned.It is also important to mention the ZT at which the light treatments were given, as this affects the photoreceptor activities c.The methods section (Lines 739-742) shows that the UVB treatment was only for 2 hours and the plants were returned to white light.Irradiation with the other wavelength is also apparently performed in a similar fashion.It is surprising that such as short treatment was able to produce the seedling phenotypes shown in Fig 2C .Again, the experimental conditions are not well explained d.It is not clear how the UVB experiments were performed for Figures 4i, 5e, 5h, 6h, Ext.data Fig. 7 and Ext.data Fig. 8 4.There are multiple problems with the protein interaction experiments a. Lane 4 and Lane 8 of the Y2H data in Fig. 3A: Seem to be duplicated b.The authors have used 3-AT presumably because the BD-GmUID1 showed autoactivation.A folddilution series should be performed in this scenario to show the interaction strength.Without this, the results can only be interpreted as a consequence of autoactivation c.Using the yeast system, the UVB-dependency of the interactions could easily be tested.I suggest the authors perform the Y2H interaction assays in a quantitative manner in the presence or absence of UVB d.Co-IP experiments were performed in Nicotiana, while the authors already have transgenic lines expressing GFP-tagged proteins.These lines could be used effectively coupled with agrobacteriuminfiltration (King et. al., 2015) for all the Co-IP and protein degradation experiments shown in the manuscript e.Moreover, the authors have produced specific antibodies that detect the Gm proteins.Then why use tagged proteins in Nicotiana?f.Equal loading controls for the input samples are missing in the Co-IP experiments g.Co-IP experiments were shown in chopped images.Hence the original blots must be provided as additional data h.In general, proper controls are missing from BiFC experiments.Ideal experiments must include positive and negative controls that improve reliability (Horstman et. al., 2014) i.The strength of interaction of GmGA2ox-like and GmUID1 under white light in Fig. 3C is much stronger than the one shown in Fig. 3H.Can the authors comment on this? j.In Fig. 3H, the method of quantification, laser intensity and exposure time used, UVB treatment and duration should be provided k. Figure 3H-3I: It is not clear why the interaction has been observed only in the cytoplasm and not in the nucleus, since GmUID1 also localizes strongly in the nucleus (Ext.Fig. 5C).Also, the subcellular localization of GmUBL1 and GmGA2ox-like was also not shown.Subcellular localization of all three proteins would help in interpreting the data shown in Fig. 3i 5. Discrepancies in the ubiquitination assay.The experiments are not convincing enough for establishing GmGA2ox-like as a substrate of GmUID1 a.The authors resort to using Nicotiana, while they have created Gm transgenic plants.Please see also comment 5a b.Fig. 4a: The misexpressed Gm proteins are shown to be ubiquitinated in Nicotiana.Are there any orthologues in Nicotiana that act as E3?If so, is it also UVB-dependent? Ideally, GmUID1 should also be co-infiltrated to test ubiquitination.Alternatively, the GFP-tagged Gm line should be used for ubiquitination assay c.Authors have used an anti-Ub antibody in Fig. 4B but infiltrated Ub-FLAG in Figure 4A.Why not use anti-Ub in both experiments?d.Fig. 4A: Blot with anti-GFP should also be shown.Loading controls are missing for the input samples.As ubiquitinated proteins are degraded while doing the experiments, MG132 is often used.It is not clear how this experiment was performed in Nicotiana e. Fig. 4B: The input samples in lanes 3 and 4 seem to be low and that may be the reason for the lower ubiquitination.f.Fig. 4C: GmUID1 seems to be auto-ubiquitinated.However, this is not addressed anywhere in the manuscript.To have more reliability, the K394R version should be included in the in vitro ubiquitination assay g.Fig. 4D: There is no explanation of the ways in which the experiment was conducted.It is not clear why the time points were shown or what to expect from this figure h.It is surprising that the GmGA2ox-like-GFP infiltrated in Nicotiana shows strong ubiquitination in Figure 4A, while it shows weak ubiquitination in Figure 4e.How would the authors explain this? i. Fig. 4F and 4G: The details of UVB experiments are missing j.Fig. 4i and 4J: Complete blots should be provided as additional data k.In Fig. 4J, blot at the end of the right-hand side, last lane: This is important data that shows the degradation of GmGA2-ox-like.However, the Ponceau staining shows that fewer proteins were loaded in this well l.Fig. 4i and 4J: Since the protein degradation is apparent from 90 minutes, the time points for the experiment should be extended up to 150 minutes, which will show the degradation dynamics.This is especially relevant since the UVB-mediated enhancement in GmUID1 could be observed only after at least 3-4 hours of UVB exposure (Fig. 5G) m.Since GmUID1 targets the D-BOX in GmGA2ox-like, a D-BOX mutated version should be used for the ubiquitination assays in order to verify that GmUID1 acts as an E3 in degrading GmGA2ox-like 6.For the UVB-induced phenotype in Fig. 2C-2H, only Gmuid1-1 is used.Is there any specific reason?Since the UVB phenotype is the basis for further experiments in the manuscript, the UVB phenotype of Gmuid1-2/Gmilpa1 and also the F1 plants resulting from the cross Gmuid1-1 x Gmuid1-2 should be used.This is required to establish the role of GmUID1 as an enhancer of plant height under UVB 7. To complement the Gmuid1-1 mutant, the authors resorted to overexpressing the full-length cDNA tagged with GFP.Ideally, the native promoter driving full-length genomic DNA should be used for complementation 8. Specific antibodies used in experiments such as the ones shown in Fig. 4 need validation experiments.The authors could easily show this via western blots using the WT, overexpression lines and mutants.What protein sizes are detected by these antibodies?Are there any non-specific bands detected?Please provide this as additional data 9.In Fig. 5A and 5B: GA51 content was not registered in the WT.Normally a basal level of GA degradation is expected in WT.Can this be explained?10.Fig. 5E: Please clarify why PAC along with GA was used in this UVB experiment 11.Fig. 6H: The difference in GUS activity seems to be rather due to different infiltration efficiency.There are no infiltration controls provided in this experiment 12.For the GUS assay of haplotypes, an ideal control could be hap5 with the INDEL.Moreover, it would be interesting to see the phenotype of hap5 in the presence or absence of UVB 13. Figure 6J: The plant height phenotype of haplotypes should be provided 14.Ext.data fig.1C and 1D: The number of replicates and the number of cells per replicate used for the experiment should be stated.Microscopy procedures should be added to the methods section Minor: 1. Lines 26-27: APC/C is abbreviated in the abstract without providing the full form 2. The effect of GA on UVB-induced hypocotyl elongation has recently been shown in Arabidopsis (Miao et. al., 2021).The results should be discussed in this context 3. Line 114: Do the authors mean the effect of UVB and GA? 4. Mostly the form "UV-B" is used in the manuscript.However, in some places "UVB" is used.I suggest making the word uniform throughout 5. Include microscopy procedures followed in the methods section 6. Lines 140-141: Do the authors mean 150 plants with WT phenotype?7. Lines 154-155: The sentence lack information on NGS used in the bulk segregant analysis 8. In Fig. 3, C, E and G could be combined with common controls 9. Line 252: The bioinformatic tool used should be specified in the methods section 10.Line 295: Space between proteasome and in Reviewer #1 (Comments for the Author): The manuscript describes the identification of a component of a ubiquitin E3 ligase from a forward genetics mutant screen for factors involvement in height determination in soybean.A mutagenized dwarfed plant was obtained from the screen and the associated gene GmUID1, identified by mapping, encodes a subunit of an APC/C-type ubiquitin ligase.A yeast 2-hybrid screen identified a gibberellin 2-oxidase-like (GA2ox) protein as a substrate for GmUID1 and evidence is presented that it is ubiquitinated by the ligase and targeted for degradation by the 26S proteasome.This reveals a novel, post-translational mechanism for the regulation of gibberellin metabolism.The dwarf phenotype occurs only in UV-B light, which stimulates the interaction of the GA2ox with GmUID1 by an unknown mechanism.Furthermore, expression of GmUID1 is promoted by UV-B.A second potential E3 ligase was identified from the yeast 2-hybrid screen and shown to interact with both GmUID1 and the GA2ox, enhancing ubiquitination of the GA2ox.Interestingly, evidence is presented for selection during soybean domestication for a GmUID1 haplotype lacking a lightresponsive element in the promoter so reducing its response to UV-B-stimulated expression and promoting reduced stature.
The work contains considerable potential novelty, but more evidence is required to support the conclusions.
1.The effect of the mutation on stature is surprisingly strong, more than I would expect from the presence or absence of a GA2ox, as there are numerous GA2ox paralogues with considerable redundancy.Can the authors be sure that the dwarf phenotype is due solely to stabilising this GA2ox rather than other GmUID1 substrates?
Response: Thank you for your suggestion.By examination of the phenotype of Gmuid1-1/GmGA2ox-like RNAi plants, our results strongly suggest that the essential role GmGA2ox-like in GmUID1-mediated plant height under UV-B irradiation.In order to estimate that the dwarf phenotype of Gmuid1-1 observed is solely due to GA2ox stabilization, we obtained the transgenic RNA interference lines of GmGA2ox-like in Gmuid1-1 (Gmuid1-1/GmGA2ox-like RNAi) in T2 generation, and grew H12, Gmuid1-1 and Gmuid1-1/GmGA2ox-like RNAi plants under white light supplemented with UV-B.We found that the height of Gmuid1-1/GmGA2ox-like RNAi plants were notably higher than that of the Gmuid1-1 (please see the new Fig.5l), indicating that GmGA2oxlike RNAi rescued the dwarf phenotype of Gmuid1-1.Our results strongly suggest that GmGA2oxlike plays an essential role in GmUID1-mediated plant elongation under UV-B irradiation, although we still could not conclude that the dwarf phenotype of Gmuid1 is solely due to GmGA2ox-like stabilization.These results have been introduced in the revised manuscript (please see the lines 364-370).
2. It is important to characterise the GA2ox and confirm its function.It is shown to be a homolog of the Arabidopsis GA2ox7, i.e. a C20-GA2ox.This should be better documented by showing a phylogenetic tree which includes the other AtGA2ox genes.Ideally its biochemical function would be demonstrated, for example using recombinant enzyme in vitro.
Response: Thank you for your valuable suggestion.We re-performed the phylogenetic tree with other AtGA2ox genes (please see the new Extended Data Fig. 6a), and found that GmGA2ox-like is close homolog of Arabidopsis GA2ox4, which belong to C19-GA2ox rather than a C20-GA2ox.This result has been added in the revised manuscript (please see the lines 208-209).
3. If it is a C20-GA2ox rather than a C19-GA2ox, then it would act early in the GA-biosynthetic pathway and GA51 levels should be lower rather than higher as shown for the Gmuid1 mutant.GA3 is not a major plant GA and is not metabolised by GA2ox.The authors need to measure other more relevant GAs, such as GA1 and GA4, as well as precursors and catabolites.
Response: Thank you for your suggestion.We measured the contents of endogenous GAs (GA1, GA4, GA20, GA9, GA8 and GA34) in H12 and Gmuid1-1 under white light alone and white light supplemented with UV-B.The results showed no difference in GAs content between H12 and Gmuid1-1 under white light (please see the new Fig.5e-j), while the gibberellin GA1, GA4, GA20 and GA9 were more abundant in the WT than in Gmuid1-1 under white light supplemented with UV-B, the non-bioactive GA8 and GA34 accumulated at a relatively high level in Gmuid1-1 relative to the H12 (please see the new Fig.5e-j).These results also provide important evidences that GmGA2ox-like belong to a C19-GA2ox.All the results have been added in the revised manuscript (please see the lines 342-355).
4. The method description for the GA analysis is inadequate and more information is required.It describes using GC-MS, but no conditions are provided.It refers to a paper in which LC-MS was used which is not helpful.
Response: Thank you so much for pointing out this problem.The method description for the GA analysis has been revised as following: To quantify the contents of GAs, H12, Gmuid1-1 seedlings were grown under white light and white light with UV-B.The stem apexes of the seedlings were collected, frozen in liquid nitrogen, and ground to a fine powder, and extracted with the traction method (methanol/water/formic acid = 15:4:1, V/V/V).The extracts were vortexed and centrifuged at 4694 × g under 4°C for 10min.The supernatants were dried by evaporation under the flow of nitrogen gas at room temperature, then dissolved in 200 μl of methanol.The sample extracts were analyzed using an LC-ESI-MS/MS system (HPLC, Shimpack UFLC SHIMADZU CBM30A system;MS, Applied Biosystems 6500 Triple), and the data were analyzed by Metware Biotechnology Co., Ltd.Wuhan, China.At least three replicates of each assay were performed (please see the lines 902-912).

Other points:
Lines 47-51: Yields were increased by using high-yielding varieties and applying high levels of fertiliser in combination with the introduction of semi-dwarfing genes to prevent lodging.The genes also improved harvest index.
Response: It has been revised as you suggested.We added the description about the harvest index, we have revised the sentences (please see the line 51).
Line 58: gibberellin rather than gibberellic acid, which is specifically GA3.
Response: Thank you for pointing out this problem.We have exchanged gibberellic acid to gibberellin in the revised manuscript (please see the line 58).

Lines 64-65: what is meant by central hub modulators?
Response: To make the sentence more concise, we have changed the description of "central hub modulators" to "Loss-of-function mutations in GA biosynthetic genes such as GA20ox and GA3ox often lead to dwarfism" (please see the lines 63-64).
Lines 163-166: the mutation modifies a splice site with the assumption that the intron is transcribed with the introduction of a stop codon.Was this confirmed by sequencing the mutant transcripts?
Response: Thank you for your suggestion.We added the sequencing results for Glyma.11G026400 in wild type and Gmuid1-1 mutant.Please see the new Extended Data Fig. 3a.
Line 326: It is not simply the balance between GA3 and GA51.Other more relevant GAs need to be analyzed.
Response: We do agree that more GAs should be analyzed.Thus, we measured the contents of endogenous GAs (GA1, GA4, GA20, GA9, GA8 and GA34) in H12 and Gmuid1-1 under white light and white light supplemented with UV-B conditions.We detail the results in our response to the Editorial comments 2 and the Result section in the revised manuscript (please see the lines 342-355).
Line 387: The HAP5 phenotype with reduced nodes and branches is not typical of lower gibberellin levels.
Response: Thank you for your suggestion.To focus on the regulation of gibberellin, we deleted the phenotypes with node numbers and branches in different haplotypes (please see the new fig.6j).apparently ubiquitinates the GA-inactivating enzyme GmGA2ox-like under UV-B, resulting in the degradation of GmGA2ox-like.This is further enhanced by another E3 ubiquitin ligase identified by the authors, namely GmUBL1.In agreement with the finding that GmUID1 ubiquitinates GmGA2ox-like, the Gmuid1 mutant shows reduced levels of active GA3 (as the catabolic GmGA2ox-like levels would be enhanced in absence of its E3 ubiquitin ligase, resulting in enhanced inactivation of active GA, and thus dwarf growth).What is not clear, however, is how these findings fit to UV-B regulation of active GA levels and growth in wild type.The work by the authors suggests that UV-B (likely by UVR8 photoreceptor signaling) should lead to elevated levels of active GA (as GmUID1 is activated, thus GmGA2ox-like levels reduced -i.e. less active GA being catabolized) and thus there should be enhanced growth under UV-B.However, available data, mainly from Arabidopsis, but also other plants, point to UVR8-dependent growth inhibition under UV-B, associated with reduced levels of GA and elevated DELLA levels.The authors should address this discrepancy.At present no data is provided how wild-type soybean responds to UV-B in the field, in regard of growth as well as GA levels.The manuscript provides potentially very interesting findings, but the below points need to be addressed (particularly also point 1) before publication.
Response: Thank you for your critical comments, which have been very helpful in improving the quality of the manuscript.

Major points:
1. UV filters should be used in the field to support and extend the findings, comparing wild type under UV and no-UV conditions (growth and GA levels under UV exclusion versus under a UV transmitting mock filter).Same for Gmuid1 mutant (i.e.rescue of phenotypes by filtering out UV in the field which would provide very strongly support that indeed UV is the underlying reason for the Gmuid1 phenotype in the field).Is UV-B indeed resulting in higher active GA levels and enhanced growth in soybean, as suggested but not investigated nor discussed in this work?If so this would be in contrast to findings in other plants, and possibly even soybean (see e.g. also Vanhaelewyn et al J. Exp.Bot. 2016, Hayes et al PNAS 2014), and thus should be well documented.Also well-controlled UV-B experiments in the glasshouse can be additionally performed.
Response: Thank you for your suggestion.We do agree that filtering out UV in the field could strongly support and extend our findings.However, it is not suitable for field cultivation currently.
To deal with this problem, we performed well-controlled UV-B experiments in the glasshouse using narrow-band UV-B as suggested here and by Reviewer 3, which was commonly used in the UV-B To further clarify the role of GmUID1-GmGA2ox-like-mediated GA catabolism in plant growth under UV-B irradiation, first we measured the contents of endogenous GAs (GA1, GA4, GA20, GA9, GA8 and GA34) in H12 and Gmuid1-1 plants under white light alone and white light supplemented with UV-B.In general, the endogenous GA content in both H12 and Gmuid1-1 plants showed a decreasing trend under white light supplemented with UV-B (please see the new Fig.5ej), which demonstrated that UV-B reduced the GA level in soybean and is consistent with the previous studies (Vanhaelewyn et al J.Exp.Bot. 2016, Hayes et al PNAS 2014).However, the reduction rate of active GA in Gmuid1-1 was higher than that in H12, indicating that GmUID1 is important in UV-B-regulated active GA levels.In addition, in consistent with the phenotype observed, there was no difference in the levels of GAs between H12 and Gmuid1-1 under white light, while the gibberellin GA1, GA4, GA20 and GA9 were more abundant in the WT plants than in Gmuid1-1 under white light supplemented with UV-B conditions, the non-bioactive GA8 and GA34 accumulated at a relatively high level in Gmuid1-1 relative to the H12 under same conditions (please see the new Fig.5e-j).Therefore, our results demonstrated that GmUID1 repressed the UV-Binduced decreasing of active GA levels to improve the UV-B tolerance in soybean.Consistently, UV-B irradiation reduced the protein levels of GmGA2ox-like in WT but not in Gmuid1-1 (please see the new Fig.4g).Together, our results suggested that GmUID1-GmGA2ox-like module enhances the UV-B tolerance in soybean by repressing UV-B-induced reduction of active GA levels.
2. The finding that UV-B promotes the interaction between GmUID1 and GmGA2ox-like should be supported by Co-IP experiments, and not be limited to BiFC experiments (Fig. 3h,i).This should be doable as Co-IPs in absence of UV-B are provided in Fig. 3b,d,f.GmUID1 -GmUBL1 and GmUBL1 -GmGA2ox-like provide control Co-IPs that should not be affected by UV-B.
Response: We agree that Co-IP should also be done to support our conclusion.Therefore, we performed the Co-IP experiments in presence or absence of UV-B.In consistent with BiFC experiments, Our Co-IP results showed that UV-B promoted the interaction between GmUID1 and GmGA2ox-like, but did not promote the interaction between GmUID1 and GmUBL1, GmUBL1 and GmGA2ox-like (please see the new Fig.3h, Extended Data Fig. 7f, 7i).
3. Clarify that GmUID1 is GmILPA1, and that the Gmuid1-2 has been published before as Gmilpa1 mutant.
Line 17: "...H12 named as Gmuid1, the other name Gmilpa1...".Not clear... Shouldn't the name previously given be kept?That would be clearer than having several names for the same gene.Is Gmuid1-2 the previously published Gmilpa1 mutant (Gao et al., Plant Physiol 2017)?Seems like from the description of the mutation (1149-bp deletion incl.4th exon...).Thus why "... we isolated additional mutant allele...".Isolated from where if already published.Please clarify.
Lines 181-182: "Sequence comparison and phylogenetic analysis demonstrated that GmUID1 encodes an AP8C-like protein, ...", rather confirmed, if at all, as the gene has already been published before (Gao et al 2017 entitled "GmILPA1, Encoding an APC8-like Protein, Controls Leaf Petiole Angle in Soybean") Line 187: "..., which revealed that GmUID1-GFP localizes to both the nucleus and cytoplasm..."; also confirmed, as already shown by Gao et al., 2017 for GmILPA1, which = GmUID1... Response: Thank you for your suggestion.We thought that GmUID1 is a UV-B-induced dwarfrelated gene, so, GmUID1 named is probably more appropriate for the main idea of this manuscript as title "UV-B irradiation-activated E3 ligase GmUID1 modulates gibberellin catabolism to increase plant height in soybean".Therefore, we didn't exchange GmUID1 to GmILPA1 in new revision.If you don't agree with us, we'll exchange GmUID1 to GmILPA1.
4. Please add western analysis of GmGA2ox-like protein levels in wild-type under UV and no-UV conditions, as well as same for Gmuid1.An anti-GmGA2ox-like antibody is available, but was only used in cell-free degradation assays (Fig. 4i,j).An effect on GmGA2ox-like levels should be tested in western analyses as well.Elevated levels in Gmuid1?Reduced levels in wild type under UV versus no-UV?Response: Thank you for your suggestion.We analyzed the GmGA2ox-like protein levels in H12 and Gmuid1-1 under UV-B and no-UV-B conditions.The results showed that the protein levels of GmGA2ox-like were similar between H12 and Gmuid1-1 under white light.However, UV-B irradiation reduced the amount of GmGA2ox-like in H12 but not in Gmuid1-1 plants, demonstrating that UV-B irradiation regulated GmGA2ox-like stabilization via GmUID1 (please see the new Fig.4g).

5) Fig 5c:
discuss why +GA only suppresses dwarfism of Gmuid1, but results in strongly changed plant morphology compared to wild type.
Response: Thank you for your suggestion.Our findings showed that GmUID1 regulated plant height under UV-B irradiation.However, we speculate that GmUID1 might be a multiple-function E3 ligase that involved in different growth and development processes via different mechanisms.For example, the Leaf petiole Angle in Gmuid1 is larger than that of H12 (Extended Data Fig. 1a, Gao et al Plant Physiol 2017), indicating that GmUID1 also controls leaf petiole angle in soybean.
Therefore, treatment with GA3, only restored plant height, but not other phenotype unrelated to GA, of Gmuid1-1 to H12.
Response: Thank you for your suggestion.We have revised as you suggested (please see the line 24).
Response: Thank you for your suggestion.We exchanged "..., effectively improving GmUID1 function."to "Our results revealed that GmUID1 mediated UV-B tolerance to maintain growth in soybean by modulating GA metabolism."(Please see lines 35-36).
Response: Thank you for your suggestion.We have removed multiple duplicates of references (please see the lines 1078-1251).
-Line 114: "affection"?Entire sentence not clear to me... Response: Thank you for your suggestion.We rephrased the sentence "Our study characterized the effect of UV-B exposure and GA application on plant height" (please see the line 113).
-Line 119/120: "... might be due to the damage of cis-regulatory elements".Rather: due to specific mutations in cis-regulatory elements?
Response: Thank you for your suggestion.We exchanged "... might be due to the damage of cisregulatory elements" to "due to the specific mutations in cis-regulatory elements" (please see the lines 118-119).
Response: Thank you for your suggestion.We added "wild type" (please see the line 139).
Response: Thank you for your suggestion.We rephrased the sentence "increased exon of the transcript" (please see the line 163).
-Fig.4c: why the smear also below the band if indicating ubiquitination?
Response: Thank you for your suggestion.We re-performed the in vitro ubiquitination assay forGmGA2ox-like-MBP.The new results clearly showed that GmGA2ox-like can be ubiquitinated (please see the new Fig.4c).
-Line 315: should likely read "... under white light in the field (Fig. 5c-d)".In general, please describe conditions clearer in figure legends.
Response: Thank you for your suggestion.We rephrased the sentence "The results showed that exogenous GA3 treatment restored the height of Gmuid1-1 to that of H12 when grown under sunlight" and added the conditions clearer in figure legends (please see lines 336-337, the new Fig.5a).
-Line 321: in contrast to statement "... within 12 h of combined treatment with GA3 and UV-B light (Fig. 5g-h)" apparently no combined treatment shown in Fig. 5g-h.
Response: We do appreciate your carefully checking and thank you for pointing out this wrong description.We rephrased the sentence "within 12 h treatment with GA3 or UV-B light" (please see the line 361).
This statement seems to refer to GUS assays in N.benthamiana... Needs to be corrected.Response: Thank you for your suggestion.It has been revised in the new Fig.2c.stronger than the one shown in Fig. 3H.Can the authors comment on this?
Response: Thank you for your suggestion.As the brightness in the Fig. 3c were adjusted, the interaction intensity between GmGA2ox-like and GmUID1 in Fig. 3c seemed much stronger under white light than that in Fig. 3h.In the revised manuscript, we used the same settings to adjust the brightness for the images in Fig. 3c  k. Figure 3H-3I: It is not clear why the interaction has been observed only in the cytoplasm and not in the nucleus, since GmUID1 also localizes strongly in the nucleus (Ext.Fig. 5C).Also, the subcellular localization of GmUBL1 and GmGA2ox-like was also not shown.Subcellular localization of all three proteins would help in interpreting the data shown in Fig. 3i Response: Thank you for your suggestion.Co-localization of GmGA2ox-like and GmUID1, GmUBL1 and GmUID1 were performed, the results indicated that GmUID1 and GmGA2ox-like, GmUID1 and GmUBL1 were co-localized in cytoplasm and nucleus by transient expression in N. benthamiana, please see the new Extended Data Fig. 8. 5. Discrepancies in the ubiquitination assay.The experiments are not convincing enough for establishing GmGA2ox-like as a substrate of GmUID1 a.The authors resort to using Nicotiana, while they have created Gm transgenic plants.Please see also comment 5a Response: Thank you for your suggestion.The GFP-tagged GmGA2ox-like lines were used for ubiquitination assay, the result showed that GmGA2ox-like can be ubiquitinated in soybean plants (please see the new Fig.4f).b.Fig. 4a: The misexpressed Gm proteins are shown to be ubiquitinated in Nicotiana.Are there any orthologues in Nicotiana that act as E3?If so, is it also UVB-dependent? Ideally, GmUID1 should also be co-infiltrated to test ubiquitination.Alternatively, the GFP-tagged Gm line should be used for ubiquitination assay Response: Thank you for your suggestion.(1) We performed ubiquitination assay by using coinfiltrated GmUID1 and GmGA2ox-like to test ubiquitination of GmGA2ox-like, the result showed that there was a remarkably increased smear representing poly-ubiquitinated GmGA2ox-like in the presence of GmUID1, please see the new Fig.4d.(2) We also performed ubiquitination assay by using the GFP-tagged GmGA2ox-like lines, the result showed that GmGA2ox-like can be ubiquitinated in soybean plants (please see the new Fig.4f).e. Fig. 4B: The input samples in lanes 3 and 4 seem to be low and that may be the reason for the lower ubiquitination.
Response: Thank you for your comments.We re-performed western blot for Fig. 4b, and the results showed that the input sample abundance of each lane was similar (please see the new Fig. 4b).f.Fig. 4C: GmUID1 seems to be auto-ubiquitinated.However, this is not addressed anywhere in the manuscript.To have more reliability, the K394R version should be included in the in vitro l.Fig. 4i and 4J: Since the protein degradation is apparent from 90 minutes, the time points for the experiment should be extended up to 150 minutes, which will show the degradation dynamics.This is especially relevant since the UVB-mediated enhancement in GmUID1 could be observed only after at least 3-4 hours of UVB exposure (Fig. 5G) Response: Thank you for your suggestion.Indeed, UVB-mediated enhancement in GmUID1 could be observed after at least 3-4 hours of UVB exposure (Fig. 5g, new Fig.5l), therefore, we tested the protein level of GmGA2ox-like in H12 and Gmuid1-1 with and without UVB, the time points were extended up to 12 hours, the result showed that GmGA2ox-like showed substantial degradation in WT exposed to UV-B from 10 h, but not in Gmuid1-1 under the same conditions, see the new Fig.

Reviewer # 2 (
Remarks to the Author): Sun et al. report the soybean mutant Gmuid1 as being dwarf specifically in the field or in the greenhouse under UV-B.GmUID1 (previously identified and named by Gao et al., 2017 as GmILPA1) is an APC8-like protein, a subunit of the E3 ubiquitin ligase APC/C.GmUID1 signaling field (Podolec, R. et al PNAS 2020; Podolec, R. et al The plant journal 2022; Yu Yang, et al Nature plants 2018).In detail, H12 and Gmuid1-1 plants were grown under white light (600 μmol m −2 s −1 , 12 h /12 h light/dark cycles) or white light supplemented with UV-B (1.5 μmol m −2 s −1 , 12 h /12 h light/dark cycles, TL20W/01RS tubes; Philips).
: n=? Field experiment?Response: Thank you for your suggestion.We added the description in figure legends."Number of GmUID1 Hap1 = 201, number of GmUID1 Hap 2 = 38, number of GmUID1 Hap5 = 81, number of GmUID1 Hap6 = 84".And The data was obtained through field statistics.
figure that one does not compare plant height in 2b versus others measurements.As they are directly next to each other, it is rather confusing.Mention the light treatments in the legend (duration, fluence rate).
and Fig.3h.j.In Fig. 3H, the method of quantification, laser intensity and exposure time used, UVB treatment and duration should be provided Response: Thank you for your suggestion.The method of quantification, laser intensity, exposure time used, UVB treatment and duration was added in the figure legends and "methods" ("The relative fluorescence intensities of cytoplasm and whole cells were quantified and the cytoplasmto-background ratios are plotted, N. benthamiana leaves were co-infiltrated with GmGA2ox-like-nYFP and GmUID1-cYFP, and exposed to 1 h of UV-B (21μmol m -2 s -1) before imaging"), please see the new Fig.3f-g and lines 928-930.
c. Authors have used an anti-Ub antibody in Fig. 4B but infiltrated Ub-FLAG in Figure 4A.Why not use anti-Ub in both experiments?Response: Thank you for your suggestion.We used anti-flag antibody in Fig.4a because Ub-Flag is a recombinant purified protein, and anti-flag antibody can also detect the ubiquitination.For Fig.4b, we used anti-Ub antibody to detect the ubiquitination level of GmGA2ox-like in Nicotiana.d.Fig. 4A: Blot with anti-GFP should also be shown.Loading controls are missing for the input samples.As ubiquitinated proteins are degraded while doing the exp Loading controls eriments, MG132 is often used.It is not clear how this experiment was performed in Nicotiana Response: Thank you for your suggestion.(1) We re-performed western blot using anti-GFP and added the results to the new Fig.4a.(2) MG132 was added in the experiment conducted by Nicotiana, and we added the description in the method of the revised manuscript (please see line 948, line 980).
you for your suggestion.(1) The related description of auto-ubiquitinated of GmUID1 was added in revised manuscript (Lines 270-271).(2) The K394R version was included in the in vitro ubiquitination assay.The result showed that GmGA2ox-like K394R cannot be ubiquitinated, please see the new Fig.4c.g.Fig. 4D: There is no explanation of the ways in which the experiment was conducted.It is not clear why the time points were shown or what to expect from this figure Response: Thank you for your suggestion.Due to the GmGA2ox-like degradation experiments have been conducted in soybeans (please see the new Fig.4i) and was repeated in Nicotiana (please see the new Fig.4j), therefore, we deleted the Fig. 4d in revised manuscript.h.It is surprising that the GmGA2ox-like-GFP infiltrated in Nicotiana shows strong ubiquitination in Figure 4A, while it shows weak ubiquitination in Figure 4e.How would the authors explain this?Response: Thank you for your comments.In Fig.4a, we used anti-flag to detect the ubiquitination level of GmGA2ox-like, In Fig.4e, we used anti-Ub antibody to detect the ubiquitination level of GmGA2ox-like.Therefore, we thought that the difference between Fig.4aand Fig.4ewas due to differences in antibodies.i. Fig.4F and 4G: The details of UVB experiments are missing Response: Thank you for your suggestion.We added the details of UVB experiments in the figure legends and methods "Immunoblots showing GmGA2ox-like protein levels in V2 stage seedlings of H12 and Gmuid1-1 grown in white light (600 μmol m −2 s −1 , 12 h/12 h light/dark) and then transferred to white light supplemented with UV-B (1.5 μmol m −2 s −1 , 12 h/12 h light/dark) for the indicated time periods" (please see the new Fig.4i).j.Fig.4i and 4J: Complete blots should be provided as additional data Response: Thank you for your suggestion.We provided all original blots in the source data.k.In Fig.4J, blot at the end of the right-hand side, last lane: This is important data that shows the degradation of GmGA2-ox-like.However, the Ponceau staining shows that fewer proteins were loaded in this well Response: Thank you for your suggestion.According to your comments, Fig.4Jhad been reperformed with the same amount of proteins loaded (please see the new Fig.4j).
4i. Fig.4iof previous manuscript was moved to the new Extended Data Fig.9.For Fig.4j, the time points were extended up to 150 minutes, the result showed that the protein degradation is apparent from 60 minutes to 150 minutes (please see the new Fig.4j).m.Since GmUID1 targets the D-BOX in GmGA2ox-like, a D-BOX mutated version should be used for the ubiquitination assays in order to verify that GmUID1 acts as an E3 in degrading GmGA2oxlike Response: Thank you for your suggestion.The D-BOX mutated version was used for the ubiquitination assays in Nicotiana, the results showed that GmUID1 cannot enhance the ubiquitination of D-BOX mutated version (see the new Extended Data Fig. 9a).